Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.

Cell division of ggeneflesmus quadricauda (Turp.) Breb. (Scenedesmaceae) is enhanced by methylglyoxal, a general inhibitor of cell division, at threshold concentration in conjunction with treatment timing related to growth stage of batch cultures. At 0.5 mM methylglyoxal concentration, cell division was significantly enhanced in algae treated in the logarithmic phase. Specific growth rates of methylglyoxal-treated cultures were rapidly increased at the beginning of logarithmic phase. Cultures inoculated with high cell numbers were less sensitive, but still showed high specific growth rates in logarithmic phase. Cell division in cultures which had low cell numbers was inhibited by 0.5 mM methylglyoxal treatment. Both specific activity of Glyoxalase I and the ratio of Glyoxalase I to Glyoxalase II of methylgloxal-treated cultures were higher than those of controls (1.3 and 2.1- fold, respectively). Pyruvate concentration in treated cultures was increased after methylglyoxal treatment.

Cytokinesis is the physical division of one cell into two driven by an actomyosin contractile ring and positioned by signals from microtubules. This process is highly regulated spatially and temporally to ensure accurate division into two daughter cells. Here, I present work that builds upon our understanding of cytokinesis, focusing on the spatial requirements for protein function during cell division and midbody assembly. In Chapter 1, I present an introduction to cytokinesis and the cell and molecular mechanisms that govern the process. In Chapter 2, I present work I contributed to on the use of Upconverting nanoparticles for co-alignment of visible and infrared light on a light microscope. In Chapter 3, I present work developing a new microscopy technology called FLIRT (Fast Local Infrared Thermogenetics) that uses infrared light to inactivate fast-acting temperature sensitive protein function with subcellular precision and validate its use to study cytokinesis and cell fate signaling in the nematode Caenorhabditis elegans. In Chapter 4, I improve upon FLIRT technology by increasing its precision and demonstrate its use in studying the spatial regulation of key cytokinesis proteins including the actomyosin cytoskeleton in contractile ring constriction. The central spindle is an array of antiparallel overlapping microtubules that forms between the separating chromosomes in anaphase and is thought to serve as a signaling hub for cytokinesis. The central spindle is thought to become compacted during contractile ring constriction to form the dense midbody at the end of cell division. In Chapter 5, I investigate the requirements for central spindle microtubules in assembling midbodies in the C. elegans one-cell embryo. I present evidence that the CENP-F-like protein HCP-1 plays a primary role relative to its paralog HCP-2 in assembling the central spindle, and that the midbody can form independently of central spindle assembly. In Chapter 6, I discuss future directions for my work on both technology development and the mechanisms of cytokinesis. Through this work, I develop new technologies and hypotheses for how cytokinesis is spatially regulated within a cell, adding new complexity to our understanding of cell division.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

In this work, we identified and characterized developmentally regulated aspects to cell division in the Xenopus laevis. We found that cells in the early neural plate divide in an oriented manner. This orientation is established by Cdc42 controlled maintenance of stable interactions between the spindle and the cell cortex. This role of Cdc42 is developmentally regulated and cells dividing later in a related tissue, the tail epidermis, are not under this control. Moreover, we find that the cell divisions in the early neural plate are further specialized in their mechanisms of cell division. Cells in the early neural plate exhibit exaggerated anaphase-B movements, a delayed onset of cytokinesis, low density of midzone microtubules and a rapid cytokinetic furrow ingression as compared to the late tail epidermis, another ectodermally derived tissue. These modifications to the mechanism of cell division appear to be because of a reduced level of PRC1, a microtubule bundling protein, and thus modifications to the central spindle structure. Finally, we find that cytokinetic mechanisms may be functionally related to the process of ciliogenesis. We find proteins known to localize to the central spindle localized to the rootlet of the basal body of cilia in multiciliated cells of the mucociliary epidermis. This localization may be related to vesicle transport during both these processes. This work reveals unexpected plasticity to fundamental mechanisms of cell division. / text

The purpose of this study was to determine the endogenous levels of indole-3-acetic acid throughout the life cycle of Chlorella pyrenoidosa, and to show a correlation between onset of cell division and IAA levels.

Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.

Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science (Biochemistry), University of the Witwatersrand, Johannesburg, 1992 / An investigation has been made into aspects of the expression of oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee). HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour lsaseccieted with hepatitis a virus (HBV) infection. c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours and adjacent hepatic tissue from the same petiEJ;htswhen compared to normal liver. Amounts of Fos and MYQ prot~in in the liver tumour specimens were else raised. The"e was some correlation between the patients' serum a..fetoproteirt concentretlons, histological features of tumour differentiatic)t"l, c..mvc and c40s r.ixpression. expression of e-tas and c..myc has been reportec to be elevated after stimulation of cells to alvlde, ,'1$ occurs during liver r19ganeration. This was corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following treatment with alkaline medium aa a mitogenlo stimulus. The time course of the expression of these oncogenes was similar to that reported after gro\l'l/th factor sttmulation, The H[~V X..gene ma\' be responsible for increased oncogene expression it' YCC as a result of its documented trans activating properties. This vi!'a~ gene is unusual in that it has a codon preferanc";which is similar to that of eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral virus to that giving rise to retroviruses. These ideas suggest that the HBV X·gene is a viral oncogene derived from a host homologue. Low stringency Northern brot hybridisation using a X-gene probe denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate the sequence from mouse heart and thymus eDNA libraries ware unsuccessful despite ext,~n$jve screening with sensitive probes (SP6 palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene \ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method " . II aimed at isolating a mammalian sequence. No sinnificant sequsnce \\ homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from \1 l! gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous gene may have resu~ted from poo' complS,JIlentarity between the VIral ant! \\ mammalian secuencec, 1\ \\ Non..~pecific amplification is commonly enct~unter&d when u$1110 PCli'. A qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an " interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts. The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe$ betwsen the internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f extension. / GR 2016

A thesis submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, South Africa, in fulfilment of the requirements for the Degree of Doctor of Philosophy, This thesis presented as a series of manuscripts 1993 / In this study the morphologic, cytokinetic, biochemical, and molecu1w: consequences of low-dose continuous Puromycin-exposure were examined in HL-60 cells, and in a variety of malignant and non-malignant human and murine cell types. Puromycin (PM) is a composite of the amino nucleoside dimethyladenosine and tyrosine-o-methylether. Functionally it is an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, more specifically of tyrosyl..tRNA. At high concentrations 5-S0#tg/ml (10-100#tM) PM has been found to block protein synthesis completely by causing the premature release from the ribosomes of truncated peptide chains which are bound to PM through their carboxyl termini. The nascent PMGpeptide complexes (PMPs) are rapidly degraded through a ubiquitin-dependent pathway and are of interest because of (i) their potential to compete for degradation with the natural substrates of the ubiquitin-dependent pathway, including cyclin B, and (ii) because their structure predicts an inhibitory effect on tyrosine kinase activity. In the current' study then, special consideration was given to the effect of PM on the cell cycle, on apoptosis (programmed. cell death), and on tyrosine kinase activity, As a means of comparison, certain of these effects were also examined with respect to another translation inhibitor Cycloheximide (CHX), to two other substituted purines Puromycin Amino nucleoside (PAN) and 6..Dimethylaminopurine (6-DMAP), as well as to the cyclophospbamide derivative Mafosfamide (ASTA Z 7557). / GR2017

A recente explosão do conhecimento da genética molecular e o avanço da indústria da biotecnologia requerem que o público compreenda muitos conceitos da genética para a tomada de decisões sobre a pertinência do uso dessas novas ferramentas. Durante os últimos 30 anos a literatura educacional produziu conceitos e teorias para lidar com estas dificuldades, mas a maioria dos professores desconhece estas produções, principalmente porque os periódicos de referência são publicados em línguas estrangeiras (principalmente em inglês). Esta pesquisa-ação pretende preencher esta lacuna e foi baseada em testes que envolveram 283 estudantes de 12 a 15 anos de idade. Nela descrevemos as concepções dos estudantes sobre a localização e transmissão da informação genética antes e após a aplicação de uma seqüência didática elaborada especificamente para desenvolver estratégias metacognitivas de aprendizagem. As idéias dos estudantes foram colhidas por meio dos questionários e redações, nas quais os estudantes descrevem como imaginam ser o interior das células e dos gametas. Verificamos que as crianças do ensino fundamental possuem concepções semelhantes àquelas descritas para estudantes do ensino médio. A comparação das redações produzidas pelos estudantes antes e após a aplicação da seqüência didática permitiu verificar que o padrão mais comum de aprendizagem é sincrético, ou seja, as crianças tendem a distorcer as informações oferecidas pelo professor em virtude da existência de conhecimentos prévios. Por fim, a descrição e a documentação de seqüências didáticas planejadas a partir de conhecimentos produzidos na literatura educacional permitem o entendimento dos processos de transposição didática e a relação deste com a aprendizagem dos estudantes. / The recent knowledge explosion on the areas of genetics, molecular biology and biotechnology introduced many new concepts hard for common people to grasp in their decision-making processes. During the last 30 years or so the educational literature produced concepts and theories to cope with these difficulties but the vast majority of our elementary and highschool teachers remain unaware of them possibly because the available literature is written in foreign languages (mainly in English). The action-research here presented intends to fill this gap and was based on tests performed with 283 students aged 12 to 15. We describe their conceptions on the location and transmission of genetic information before and after the application of a didactic sequence specifically elaborated to develop metacognitive learning strategies. The students\' ideas were gathered by means of questionnaires and through essays describing how they imagine the interior of cells and germ-cells. We verified that children on basic educational level have conceptions very similar to those of students of middle-level education. The paired comparison of before and after essays suggests the existence of a common, syncretic learning standard. In plain language, the results indicate that previous informal knowledge of children tends to distort the formal information transmitted by their teachers. It is clear that the description and documentation of planned didactic sequences, available from the specialized literature, provide the understanding of the didactic transposition process and its relation with the students´ learning process.